A “toehold hairpin RNA” sensor is a key to the process. If a single strand of RNA includes complementary sequences at separated stretches along its length, it can fold back upon itself to form a hairpin. The Wyss researchers engineer an RNA sequence so that it includes: a stretch of detector RNA that will bind to messenger RNA produced by the target (a transcript Ebola virus produces to build coat proteins for new viruses, for example); a ribosome binding site sequence, which will prompt the ribosome to grab the molecule and start reading its instructions to make protein; a “closure” sequence that binds to the detector RNA, hiding the ribosome binding site in the loop of the hairpin; and mRNA instructions for an enzyme (such as beta-galactosidase) that will alter the structure of a reporter molecule (such as a yellow form of galactose) to change its color (say, to a purple).
The design leaves the toehold, a short strip of detector RNA, dangling free at the bottom of the hairpin. The target RNA latches onto the toehold and starts zipping up along the rest of the detector sequence—and unzipping the closure sequence. This opens up the ribosome binding site in the hairpin. The unfolded mRNA then passes through the ribosome, and the ribosome produces the enzyme. The enzyme reacts with the reporter and, voila, the color changes.
Cost. Paper-based diagnostics could cost as little as US $0.02 to $0.04 per sensor, they say. This is dramatically lower than the $0.45 to $1.40 for familiar antibody-based rapid diagnostics tests (RDTs) like home pregnancy and glucose kits, and the $1.50 to $4.00 cost of the reagents used in a PCR (polymerase chain reaction) DNA assay.
Speed. The paper-based synthetic gene network diagnostics the Harvard team produced are about as fast as antibody-based home tests, a little faster than PCR, and much faster than the bacterial and viral culture methods that have been a diagnostic mainstay. The Wyss group’s paper diagnostics produce detectable color changes in 20 to 40 minutes (or perhaps longer, depending on the assay and the concentration of the target molecule). Antibody-based RDTs show results in about 20 minutes. And PCR assays require at least 60 minutes…and a well-equipped laboratory.
via Printed Dots Detect Ebola (and More) Without a Lab – IEEE Spectrum.
Paper-Based Synthetic Gene Networks
Pardee, Keith et al.
Cell , Volume 159 , Issue 4 , 940 – 954
Abstract:
Synthetic gene networks have wide-ranging uses in reprogramming and rewiring organisms. To date, there has not been a way to harness the vast potential of these networks beyond the constraints of a laboratory or in vivo environment. Here, we present an in vitro paper-based platform that provides an alternate, versatile venue for synthetic biologists to operate and a much-needed medium for the safe deployment of engineered gene circuits beyond the lab. Commercially available cell-free systems are freeze dried onto paper, enabling the inexpensive, sterile, and abiotic distribution of synthetic-biology-based technologies for the clinic, global health, industry, research, and education. For field use, we create circuits with colorimetric outputs for detection by eye and fabricate a low-cost, electronic optical interface. We demonstrate this technology with small-molecule and RNA actuation of genetic switches, rapid prototyping of complex gene circuits, and programmable in vitro diagnostics, including glucose sensors and strain-specific Ebola virus sensors.